DE10313035A1 - Method to increase the apoptotic effect of cytostatics without increasing toxic side effects - Google Patents

Abstract

The invention relates to the use of at least one overexpression inhibitor of DNA repair genes and oncogenes for producing a drug to increase the apoptotic effect of cytostatics after chemotherapy.

Description

The invention consists of a method the sensitivity of tumors opposite Cytostatics over to get a long treatment period. Here, at the same time non-specific toxic side effects decreased.

• 1. auf der Hemmung der Zytostatika induzierten Überexpression des Zellproliferationsgens DDX1
• 2. auf der Hemmung der Zytostatika induzierten Überexpression von DNA-Reparaturgenen (UBE2N oder APEX),
• 3. auf der Hemmung der Zytostatika induzierten Überexpression von Onkogenen (STAT3 oder JUN-D),
• 4. auf der Hochregulierung von mikrofilament- oder mikrofilamentregulatorischen Proteinen (Actin, Tubulin, Myosin oder Tropo-Modulin),
• 5. auf der Herunterregulierung von Proteinen, die in ATP Generierung involviert sind (Succinat Dehydrogenase oder Pyruvat Dehydrogenase).

The sensitivity is maintained by maintaining and enhancing the apoptotic effect of cytostatics. This effect is based

• 1. on the inhibition of cytostatics-induced overexpression of the cell proliferation gene DDX1
• 2. on the inhibition of cytostatics-induced overexpression of DNA repair genes (UBE2N or APEX),
• 3. on the inhibition of cytostatics-induced overexpression of oncogenes (STAT3 or JUN-D),
• 4. on the upregulation of microfilament or microfilament regulatory proteins (actin, tubulin, myosin or tropo-modulin),
• 5. Down regulation of proteins involved in ATP generation (succinate dehydrogenase or pyruvate dehydrogenase).

The reduction of toxic side effects is through the simultaneous increase of DT diaphorase activity reached.

The effect is achieved by the following treatment scheme. First, a base analog is administered in parallel to that of a cytostatic. This treatment corresponds to that in the patent EP 0 806 956 B1 "Use of 5'-substituted nucleosides for the inhibition of resistance formation in the cytostatic treatment". This treatment inhibits the formation of gene amplification. After the chemotherapy, in the recovery phase ("recovery phase" after the end of the co-treatment), the treatment is carried out with the sole administration of a base analog continued. This treatment method, with which the treatment success is decisively improved, is the subject of the present invention.

It was discovered by accident that the effect of 5'-substituted Nucleosides, i.e. of base analogs, thereby significantly strengthening that the base analogue continues after stopping the cytostatic treatment is applied. In this "recovery phase" certain base analogs practice a strong effect on the expression of cancer relevant Genes from. This effect goes hand in hand with a comparison to the pure one Enhanced cytostatic treatment Apoptosis. For given alone, the base analogues did not induce apoptosis. A synergistic effect is therefore ruled out.

Apoptosis is a form of programmed Cell death. In contrast to necrosis, apoptosis is physiological Form of cell death. Based on differences between necrosis and Apoptosis can be differentiated between these two forms of cell death. Apoptosis has defined morphological and biochemical characteristics, which occur one after the other as events in an ordered cascade. The continuous process leaves divide into phases. Morphological characteristics of apoptosis are nuclear chromatin condensation (karyopyknosis), shrinkage of the Cytoplasm, formation of apoptotic vesicles and finally apoptotic Body.

The effect of cytostatics is based mainly on the stimulation of apoptosis. An integral part of the Invention, the decrease in apoptotic due to resistance formation To prevent or at least delay the effect.

Cancer is in humans one of the most common Causes of death and chemotherapy is the most common treatment. The insufficient chances of recovery from chemotherapy are due to the emergence of resistance. These resistances have their cause in that cytostatics influence the expression of genes and have a genotoxic effect, i.e. mutations, gene amplifications and recombinations induce and thus unstabilize the genome. In this way induces or selects chemotherapy resistant cancer cells. Of such effects induced by cytostatics are often oncogenes such as. Ras, Bcl2, Bcrabl, Myc, Erb82, and others affected. to Chemoresistance also the misregulated expression of genes that have something to do with DNA repair and recombination have to do with (e.g. p53 gene, BRCA1 / 2, UBE2N, APEX and Rad51). Enzymes that metabolize cytostatics and bioactivators (e.g. DHFR, DT-diaphorase (DT-D), or proteins, transport the cytostatics (e.g. MDR1).

Most cytostatics eliminate tumor cells by inducing apoptosis. Tumor cells can prevent this by over-activating survival mechanisms. Mechanisms of chemoresistance also include genes with an anti-apoptotic effect, such as STAT3, the activated “signal transdu” cer and activator of transcription 3 "or JUN-D.

invention

In 1995, previously unknown effects of certain hormones and 5-substituted nucleosides were discovered. They suppressed 2-amino-6-mercaptopurine (AMP) -induced SV40 amplification in Chinese hamster cells, and triethylene melamine-induced recombination in yeast. The discovery that treatment of mouse leukemia cells with 5-substituted nucleosides inhibits doxorubicin (adriamycin) -induced Mdr1 gene amplification and chemoresistance led to the patent EP 0 806 956 B1 ,

In the In vitro studies were 5-substituted nucleosides (i.e. base analogs) always applied together with one or more cytostatics. Fortuitously could the cells at the end of an experiment with the base analog alone BVDU continue to grow. To our surprise the BVDU effect had increased, instead of easing. Thus, as shown in Example 1, following new situation: base analogs, given on their own, none Effect of base analogs, given together with a cytostatic agent, Effect of base analogs, for themselves given alone after having previously taken it with a cytostatic had been given (recovery phase), increased effect.

• 1. Durch Blockade von "survival pathways" in der Erholungsphase (recovery).
• 2. Durch Blockade von DNA-Reparatur assoziierten Enzymen.
• 3. Durch Induktion von DT-Diaphorase Aktivität.
• 4. Durch reduzierte Expression ATP-generierender Enzyme in der Erholungsphase.

The effect, ie the inhibition of chemoresistance and increased chemosensitivity, can be described as an atoxic maintenance of cytostatics-induced apoptosis by influencing the gene expression of certain genes. this happens

• 1. By blocking "survival pathways" in the recovery phase (recovery).
• 2. Enzymes associated by blocking DNA repair.
• 3. By induction of DT diaphorase activity.
• 4. By reduced expression of ATP-generating enzymes in the recovery phase.

To 1) Base analogs like BVDU block "survival pathways" primarily in the Recovery phase of the co-treatment after stopping the cytostatics and thereby enforce the course of apoptosis.

Using HOPI double staining from AH13r tumor cells from the rat could be shown to have cytostatics such as doxorubicin (DOX), mitoxantron (MXA) or mitomycin C (MMC) Trigger apoptosis. Co-treatment with the base analogue (E) -5- (2-bromovinyl) -2'-deoxyuridine (BVDU) promotes the Apoptosis by blocking antiapoptotic "survival pathways", the STAT3 and JUN-D include.

This effect only occurs in the recovery phase (recovery phase) of the cells, as can be seen in Example 2.

Stat3 is constitutively activated oncogene and carries to the development of various human cancers. This is done by inhibiting apoptosis. Easier this way STAT3 survival of tumor cells and makes cells resistant to chemotherapy. Accordingly induced the inhibition of "Stat3-signaling" apoptosis and increases sensitivity to cytostatics.

Jun-D, a member of the Jun gene family, is an essential component of the "activating protein-1" (AP-1) transcription factor complex with ubiquitous expressiveness. Jun-D ^{(- / -)} primary fibroblasts show premature aging and increased apoptosis UV radiation or TNFα treatment. This result suggests that JUN-D activates the "NFkappaB survival pathway". In addition, p202, which is directly regulated by JUN-D, makes fibroblasts more resistant to apoptosis.

Co-treatment by BVDU reduced the expression of both JUN-D isoforms by about a quarter. In contrast was STAT3 in the recovery phase by about two thirds down regulated. (Example 2).

The effect is particularly impressive in the recovery phase after co-treatment with mitomycin C. Reduced here the base analogue overexpression of the oncogene JUN-D to the control level (example 2).

To 2) block base analogs like BVDU DDX1. DDX1 is coamplified with MYCN and overexpressed in neuroblastoma (NB) and retinoblastoma cell lines and tumors. NB patients with Amplification of both DDX1 and MYCN are worse Prognosis as patients with MYCN gene amplification only. DDX1 has therefore oncogenic potential.

Co-treatment of MMC with BVDU reduced the overexpression of UBE2N and APEX by about two thirds. Modifications from UBE2N affect resistance to DNA damage .. APEX Nuclease is a DNA repair enzyme. Blockade of APEX expression doubled cell lysis and increased DNA breaks.

To 3) BVDU induces DT diaphorase (Example 3). This has two important properties for chemotherapy. On the one hand, it activates cytostatics from the quinone and class on the other hand reduces non-specific toxic effects on the Formation of reactive oxygen species are based.

The failure of the DT-D gene leads to myeloid hyperplasia and reduced apoptosis rates due to reduced p53 and p73 expression. This is consistent with the observation that a multifactorial "multidrug resistance" phenotype of tumor cells decreases and does not increase DT-Di aphorase expression includes. Interestingly, the DT-D enzyme activity also stabilizes the lymphocyte populations. This effect could have a beneficial effect on stabilizing the patient's immune system during chemotherapy.

Many cytostatics such as DOX and MXA, upset the redox status and mitochondrial respiration of the cancer cell. This leads to Generation of reactive oxygen species (ROS). Affected by the sudden accumulation ROS is not only the cancer cell, but also all other cells, causing unwanted Side effects occur with chemotherapy.

DT-D inactivates ROS and protects cells before unspecific ROS and electrophilic attacks. As an indication of this Effect of BVDU on reducing unwanted side effects Chemotherapy in Example 4 is the weight gain of doxorubicin + BVDU-treated rats listed. DOX alone treatment leads because of the toxic side effects to weight loss. That's for sure, that BVDU only causes side effects (possibly those characteristic of DOX cardiotoxicity) be reduced, but not the toxic effects on the Tumor, To 4) Modified Expression of various enzymes in the recovery phase becomes the cytostatic Maintain effect even in the absence of a cytostatic. As can be seen in Example 5, the expression of eight genes elevated, that is degraded by six genes.

The gene products influence the Formation of microfilaments, differentiation, signal transduction and the ATP generation.

Example 1:

BVDU treatment increases sensitivity of AH13r sarcoma cells Chemotherapy-induced apoptosis. This effect remains Discontinuation of the cytostatic in the so-called Recovery phase consist.

AH13r cells were increasing doses of the cytostatic Mitomycin C (MMC) exposed. BVDU, alone given, showed no toxic effect. MMC + BVDU treatment followed three treatment cycles to reduce the number of cells in comparison for treatment with MMC alone. This inhibitory effect persisted Discontinue the cytostatic in the next Cycle in which Recovery phase exist. The cells without MMC and BVDU continued to grow unchecked. However, those who who continued to receive BVDU were strongly inhibited in their growth.

Corresponding results were with Methotrexate (MTX), doxorubicin (DOX) and mitoxantrone (MXA) too achieved when using human tumor cells.

Evidence that the reduction the cell count based on apoptosis was determined using Hoechst 33258 / propidium iodide (Hopi) double staining detected (Grusch et al., 2001).

Example 2:

We examined various “survival pathways "by means of Western blot analysis. The analyzes were carried out according to standard methods carried out (Sambrook & Russell, 2001). antibody dilutions: P-STAT3 (Cell Signaling) 1: 500, JUN-D (Santa Cruz, California) 1: 1,000. The upper of the two JUN-D bands shows the "full length isoform" and the lower band shows the "truncated isoform", which is 48 amino acids shorter. Both isoforms can activate the transcription, but the "full length" variant is more effective than the "truncated" isoform.

The content determined by densitometry on oncogene proteins JUN-D and STAT3 was in after DOX treatment the recovery phase (r = recovery phase) by a quarter or two Third reduced. In the "recovery" only. BVDU is given, no cytostatic.

A corresponding result was achieved in the investigations with Mitoxantron (MXA).

In the experiment with Mitomycin C (MMC) BVDU, given in the "recovery", completely inhibited the MMC induced JUN-D overexpression to the control level.

Example 3:

Measurement of DT diaphorase (DT-D) was carried out as dicoumarol-inhibitable NAD (P) H: dichlorophenolindophenol Reductase, as recently described (Hodnick & Sartorelli, 1997). We examined extracts from an equal number of cells, who had been treated with DOX +/- BVDU for DT-D activity. With BVDU treated cells showed approximately three times the DT-D activity than that Cells from the control or DOX alone group.

Corresponding results were obtained with Mitoxantron (MXA) and methotrexate (MTX) achieved. BVDU alone increases the DT-D activity constant, but sometimes weak.

The results with methotrexate (MTX) and human K562 tumor cells are shown in the figure above. MB means MTX + BVDU. Passage means dilution and conversion of the cells for further growth. The relative DT-D activity is recorded on the Y axis.

Example 4:

Reducing toxic side effects of doxorubicin (DOX) could be shown in the experiment with rats. SD rats were treated with dimethybenzanthrances (DMBA). The hereby induced tumors were growth-induced by DOX treatment (1 mg / kg) inhibited. While treatment and one day after each treatment, i.e. in the The animals received a recovery phase of 15 mg / kg each BVDU.

Example 5:

Collection of through treatment with base analogs and mitomycin C-influenced proteins. The results the implementation two-dimensional gel electrophoresis are as follows Table summarized.

references

• Grusch, M., Fritzer-Szekeres, M., Fuhrmann, G., Rosenberger, G., Luxbacher, C., Elford, H.L., Smid, K., Peters, G.J., Szekeres, T., & Krupitza, G. 2001. Activation of caspases and induction of apoptosis by novel ribonucleotide reductase inhibitors amidox and didox. Exp Hematol, 29 (5): 623-632.
• Hodnick, W.F. & Sartorelli, A.C. 1997. Measurement of dicumarol-sensitive NADPH: (menadione-cytochrome c) oxidoreductase activity results in an artifactual assay of DT diaphorase in cell sonicates. Anal Biochem, 252 (1): 165-168.
• Sambrook, J. & Russell, D.W. 2001. Molecular Cloning (3rd ed.). Cold Sprig Harbor, New York: Cold Spring Harbor Laboratory Press.

Claims (8)

Method for enhancing the apoptotic effect of cytostatics without increasing toxic side effects by a) inhibiting the overexpression of cell proliferation genes such as DDX1, b) inhibiting the overexpression of DNA repair genes such as UBE2N or APEX, c) inhibiting the overexpression of oncogenes such as STAT3 or JUN-D , d) upregulation of microfilament or microfilament regulatory proteins such as actin, tubulin, myosin or tropomoduline, e) downregulation of proteins involved in ATP generation, such as succinate dehydrogenase or pyruvate dehydrogenase, f) Up-regulation of the DT diaphorase. Use of base analogues after chemotherapy in the recovery phase. Use of BVDU after chemotherapy in the Recovery period. Use of base analogs as a co-treatment in of chemotherapy. Use of BVDU as a co-treatment in chemotherapy. Use of co-treatment in chemotherapy, where chemotherapy is defined as treatment with or any number of cytostatics. Use of base analogs in a concentration which can achieve a blood level of 0.02 to 50 μg / ml. Use of BVDU in a concentration that a Blood levels from 0.02 to 50 μg / ml can be achieved.